In general, the present invention relates to the field of plant molecular biology. Specifically, the present invention relates to transgenic plants with inserted transgenes that are activated by development-specific promoters.
Leaf senescence is a phase of development during which cells undergo distinct metabolic and structural changes prior to cell death (Noodxc3xa9n, Senescence and Aging in Plants, (L. D. Noodxc3xa9n and A. C. Leopold, Ed.), pp. 391-439, Academic Press, San Diego, Calif., 1988). It is an important phase in the plant life cycle that is thought to contribute to fitness by recycling nutrients to actively growing regions. The initiation of leaf senescence can be induced by a variety of external factors such as shading, mineral deficiency, drought and pathogen infection (Thomas, et al., Ann. Rev. Plant Physiol. 31:83-111, 1980) and by developmental processes such as seed development (Noodxc3xa9n, 1988, supra). In the absence of such factors, leaf senescence occurs in an age-dependent manner in many species (Batt, et al., J. Exp. Bot. 26:569-579, 1975; Hensel, et al., Plant Cell 5:553-564, 1993; Jiang, et al., Plant Physiol. 101:105-112, 1993).
Physiological and genetic studies indicate that senescence is a highly regulated process (Noodxc3xa9n, 1988, supra; Thomas, 1980, supra). The progression of a leaf through the senescence program is visibly marked by the loss of chlorophyll and consequent yellowing, a result of the disassembly of the chloroplast (Thomson, et al., Plant Senescence: Its Biochemistry and Physiology, pp. 20-30, 1987; Woolhouse, Can. J. Bot. 62:2934-2942, 1984). Leaf senescence involves degradation of proteins, nucleic acids and membranes, and the subsequent transport of the nutrients resulting from this degradation to other regions of the plant, such as developing seeds, leaves, or storage organs (Noodxc3xa9n, 1988, supra; Woolhouse, 1984, supra).
Molecular studies indicate that changes in gene expression are associated with the senescence program. The levels of mRNAs encoding proteins involved in photosynthesis decrease during senescence (Bate, et al., J. Exp. Bot. 42:801-811, 1991; Hensel, et al., Plant Cell 5:553-564, 1993; Jiang, et al., Plant Physiol. 101:105-112, 1993), while mRNA levels of genes encoding proteins thought to be involved in the senescence program increase (Graham, et al., Plant Cell 4:349-357, 1992, Hensel, et al., Plant Cell 5:553-564, 1993; Kamachi, et al., Plant Physiol. 93:1323-1329, 1992; Taylor, et al., Proc. Natl. Acad. Sci. USA 90:5118-5122, 1993). The activities of several enzymes that are likely to play a role in the breakdown and mobilization of nutrients have also been shown to increase during senescence (Blank, et al., Plant Physiol. 97:1409-1413, 1991; Debellis, et al., Plant Cell Physiol. 32:1227-1235, 1991; Friedrich, et al., Plant Physiol. 65:1103-1107, 1980; Pistelli, et al., J. Plant Physiol. 19:723-729, 1992).
Although the general changes that occur during senescence are known, many of the biochemical details of how nutrient remobilization occurs remain to be determined. Furthermore, little is understood of how the changes in gene expression that accompany senescence are regulated.
Promoters capable of promoting gene expression during the plant developmental stage of senescence are needed in the art of plant molecular biology.
As a first step towards obtaining this goal, we investigated macromolecular changes that occur during leaf senescence in Arabidopsis thaliana. The onset of leaf senescence in Arabidopsis is determined by leaf age (Hensel, et al., supra). This predictability of the senescence program in Arabidopsis facilitated an integrated study of changes in RNA, chlorophyll, protein, and gene expression associated with natural leaf senescence in the intact plant. We also used this system, as recited here, to isolate and characterize the temporal expression patterns of mRNAs that increase and decrease in abundance during leaf senescence. These senescence-specific mRNAs allowed us, as described below, to isolate and characterize novel senescence-specific promoters.
The present invention is a genetic construct comprising an SAG12 promoter sequence operably connected to a protein-coding DNA sequence not natively connected to the promoter sequence. Preferably, the SAG12 promoter sequence is the SAG12-1 sequence. Most preferably, the SAG12 promoter is the first 602 bp of SEQ ID NO:2 and the protein-coding DNA sequence encodes isopentenyl transferase.
The present invention is also a cell or a plant containing the genetic construct.
It is an object of the present invention to provide a genetic construct with a promoter sequence enabling senescence-specific gene expression operably linked to a protein-coding sequence.
It is another object of the present invention to provide a senescence-specific promoter linked to a sequence encoding an enzyme that catalyzes the synthesis of a plant hormone, preferably cytokinin.
It is another object of the present invention to provide a senescence-specific promoter linked to an isopentenyl transferase sequence.
It is another object of the present invention to provide a transgenic plant that contains a transgene expressed only in senescing tissue.
It is a feature of the present invention that gene expression can be targeted specifically to senescing tissue, thus avoiding constitutive expression that could be damaging.
Other objects, advantages, and features of the present invention will become apparent after review of the specification, drawings, and claims.